1. Purification of BamH1 endonuclease from B. amiloliquefaciens -
The final enzyme is expected to be of commercial grade free of other
nucleases and of phosphatase. Each group is expected to purify the
enzyme starting from cells grown in 4 liters of medium. The students
are exposed to the following techniques.

-cultivation of bacteria
-lysis of cells by sonic disruption and use of French Press
-salt fractionation of proteins
-separation of proteins on ion exchange and hydroxylapatite
columns
-assay of endonuclease activity
-determination of specific activity
-electrophoretic separation of DNA fragments on agarose
-test for extraneous nuclease
-test for ligatability of fragments generated by the purified enzyme

2. Purification of plasmid DNA from E. coli. Each group
is expected to purify plasmid DNA to be used for the preparation of
DNA size standards. Transformants harboring different plasmids are
grown on a 2 liter scale and plasmid DNA is isolated and ultimately
purified on CsCl. During the course of this exercise students learn
the following techniques.

-conversion of cells to protoplasts
-extraction of nucleic acids free of protein
-precitation of nucleic acids
-purification of DNA on CsCl gradients
-quantitation of DNA by absorbance at 260 nm
-large-scale digestion and recovery of DNA

Projects assigned to individual groups

1. Preparation of DNA size standards. One group of students is
assigned to collected the different plasmid digested to yield the
desired fragment sizes. The students are expected to test the
concentrationsof the different plasmid digests and to mix them in
proportion that will achieve a well balanced mixture with fragment
sizes ranging from 15 kb to 0.1 kb.

2. Construction of plasmids for expression of yeast genes from
GAL4 promoter. Each of two groups are constructing two plasmids.
One set of plasmids will have the LEU2 gene as a selectable marker in
yeast, the second set of plasmids will have URA3 as the marker. The
plasmids will also differ in the orientation of the GAL4 promoter
region with respect to the multiple cloning region derived from
pUC18. The students will learn the following methods.

-design of primers and PCR amplification DNA with new restriction
sites
-separation and recovery of DNA fragments from preparative agarose
gels
-ligation of DNA fragments
-transformation of E. coli with ligation mixtures
-plasmid miniprep screening of transformants
-partial digestion of plasmids
-destruction of restriction sites by Klenow filling-in of protruding
ends
-yeast transformation
-galacatose induction in yeast of test gene cloned into the different
GAL4 plasmids

3. Cloning and expression of Taq polymerase. This project is
assigned to four groups. Two groups will be cloning the
amino-terminal coding region of the polymerase by PCR amplification
such that the fragment can be inserted into a high-expression E.
coli plasmid. The other two groups will be cloning the rest of
the coding region by preparing a recombinant library and screening it
by colony hybridization. This will minimize the chances of
introducing mutations into the active region of the enzyme. In
addition to some of the methods and concepts described above this
project will expose the students to the following methods.